Engineering mRNA Stability for Coordinated Expression of Multiple Genes in New Operons

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The goal of this project is to develop the experimental methodologies to engineer synthetic novel operons for coordinated expression of multiple genes in bacteria. The specific aims are: (1) to develop a dual-gene expression system that allows for convenient insertion of hairpins, RNase cleavage sites, and genes; (2) to design synthetic hairpins to be used to stabilize one or more coding regions of an operon; (3) to confirm that the synthetic inserts form the structures predicted by RNA folding algorithms; (4) to test the effects of hairpin strength and endoribonuclease cleavage site location, and gene location in an operon on mRNA stability and protein production; (5) to test the effect of RNase E sites in the genes and RNase III sites in the hairpins on the stability of the transcript for these genes; and (6) to test the synthetic operon with a dual-enzyme metabolic pathway that has an easily assayable intermediate.